The Prevalence of Carbon-13 in Respiratory Carbon Dioxide As an Indicator of the Type of Endogenous Substrate The changefrom lipid to carbohydrate during the respiratory rise in potato slices Downloaded from BRUCE S. JACOBSON, BRUCE N. SMITH, SAMUEL EPSTEIN,* and GEORGE G. LATIES From the Department of Botanical Sciences and Molecular Biology Institute, University of California at Los Angeles 90024, and the Division of Geological Sciences, California Institute of Technology, Pasadena 91109. Dr. Smith's present address is the Department of Botany, University of Texas, Austin 78712. www.jgp.org ABSTRACT Isotope discrimination is a common feature of biosynthesis.in nature, with the result that different classes of carbon compounds frequently on August 22, 2006 display different IsC/isC ratios. The 18C/iSC ratio of lipid in potato tuber tissue is considerably lower than that for starch or protein. We have collected respiratory COs from potato discs in successive periods through 94 hr from the time of cutting--an interval in which the respiration rate rises 3-5-fold. The 18C/XSC ratio of the evolved COs was determined for each period, and com- pared with the 13C/ISC ratios of the major tissue metabolites. In the first hours the carbon isotope ratio of the COs matches that of lipid. With time, the ratio approaches that typical of starch or protein. An estimation has been made of the contribution of lipid and carbohydrate to the total respiration at each junc- ture. In connection with additional observations, it was deduced that the basal, or initial, respiration represents lipid metabolism--possibly the a-oxida- tion of long chain fatty acids---while the developed repiratlon represents con- ventional tricarboxylic acid cycle oxidation of the products of carbohydrate glycolysis. The true isotopic composition of the respiratory CO2 may be ob- scured by fractionation attending the refixation of COs during respiration, and by CO,. arising from dissolved COs and bicarbonate preexisting in the tuber. Means are described for coping with both pitfalls. INTRODUCTION Plant tissues are frequently characterized by a reserve of substrate, a con- dition which is particularly true for a classical object of plant respiration The Journal of General Physiology THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 55 " I970 study, the storage organ slice. Such tissues do not respond markedly to exogenous substrate. Reserve metabolites are frequently of more than one kind, and it is a matter of some interest to identify the actual endogenous substrate. Respiration in plant tissues may comprise more than one respira- tory path. The matter is further complicated by evidence that the relative contribution of coexisting respiratory paths may change with time, as is the case for the sigmoidally increasing respiratory activity of potato discs aerated for 24 hr (1). One piece of evidence pointing to changes in respiratory quality is the concomitant development of malonate sensitivity and cyanide resistance during the aging period (2--4). In addition, aged tissue readily oxidizes glu- cose and Krebs cycle acids, as measured by l~COs release from exogenously provided labeled compounds, while fresh tissue totally lacks this ability (5). To date the only intimation of the possible nature of the carbon path com- prising the basal respiration has been the evolution of 14CO2 by fresh slices Downloaded from from long-chain, carboxyl-labeled fatty acids. The characteristics of the latter degradation point to a-oxidation (6). Since the ability to degrade an exoge- nous carbon source does not necessarily reveal the nature of the endogenous respiratory substrate, we have sought to identify the endogenous substrate by comparing the I~C/~sC ratio in the normal respiratory COs with the 1~C/12C www.jgp.org ratio in the various classes of endogenous compounds. The analysis is based on the fact that various classes of tissue metabolites are characterized by their natural relative 13C/~sC ratios. on August 22, 2006 Different classes of substrates (e.g. amino acids and lipids) vary from one another in ~3C/12C ratio as a result of isotopic fractionation during biosyn- thesis. Extensive fractionation by plant tissue occurs in photosynthetic fixation of carbon dioxide (7), and in dark fixation as well (see below). Such fractionation is indicated by the markedly larger 13C/12C ratio of atmospheric COs than of the total plant carbon (7, 8). To a lesser degree, carbon isotope fractionation occurs in the conversion of carbohydrate to lipid (9, 10), and lesser still during the synthesis of cell wall material (10) and amino acids (9). Altogether, isotopic fractionation during biosynthesis results in different ~3C/1~C ratios in the various classes of metabolites. It is these differences which permit us to determine which substrate is utilized by respiring potato discs during any stage of disc aging. The procedure first requires determination of the ~nC/~2C ratios of the classes of endogenous metabolites which are potential substrates for potato tuber tissue respiration. As respiration of slices proceeds over a 24 hr period, COs is periodically collected, and the 18C/asC ratio therein is determined. The metabolite which most nearly resembles the respiratory COs in ~3C/~sC ratio at a given time is taken to represent the predominant respiratory sub- strate at that time. Though there have been suggestions regarding the nature of the respira- B. S. JACOI~ON ]ST AL. Prevalenceof ~IC in Respiratory C02 tory substrates in aging potato slices from labeling and inhibitor studies (1, 3, 4), direct evidence is lacking. On the basis of a comparison of the IaC/nC ratios of respiratory C02 through a 24 hr aging period with the ratios of endogenous metabolites, we have been able to deduce that the respiratory substrate is initially lipid and gives way to starch upon aging. MATERIALS AND METHODS Tissue discs 1.1 mm thick and 9 mm in diameter were prepared from Idaho Russet Burbank potatoes. Halved tubers were pierced within the vascular ring with a stain- less steel cork borer, the core remaining in the tuber. The tuber halves were then sliced with a microtome blade fixed in an appropriate bed (l l) to yield discs. At least 100 g of discs washed with 12 liters of CO~-free water were readied for use in 30 rain. Slices were prepared at 2°C. After slicing, 50-75 g of potato discs were aged in a 1 liter flask containing 200 ml of CO2-free l0 -4 M CaSO4. The bathing solution was changed four or five times dur- Downloaded from ing the 24 hr aging period. The pH of the solution was maintained at the desired value with an automatic titrator. The electrodes and titrant delivery tube were in- serted into the bathing solution through a rubber stopper held in a hole in the side of the incubation flask. A pH of 5.0 was maintained with 0.4 N H~SO4, and a pH of 7.2 with 0.4 M Tris. www.jgp.org A rubber stopper in the top of the incubation flask held the air inlet and outlet tubes. The outlet tube led to a liquid nitrogen trap, where CO~ evolved from the tis- sue was collected (7). Air entering the flask was scrubbed free of CO2 by passage through two 3 ft columns, the first containing Drierite, a desiccant, and the second on August 22, 2006 Ascarite, a CO2 absorbent. The flow of air through the system was 1 l0 ml/min. Extraction Procedures Though starch is the predominant food reserve in potato tuber, possible respiratory substrates such as lipids and protein were also isolated and analyzed for their laCp~C ratios. Starch was extracted either by centrifugation of starch granules from a slurry of homogenized potato tuber or by formation of an aqueous starch gel. Starch was first gelled by extracting the tissue with hot 80% ethanol. The starch-containing residue was then boiled in water and centrifuged. The supernatant contained aqueous starch gel. The two methods yielded starch with identical ~C/12C ratios, indicating there was no isotopic carbon exchange between starch and hot alcohol. Lipid was extracted from air-dried potato tissue with chloro- form-methanol (2: l, v/v). Nonlipid material was extracted from the lipid-contain- ing solvent with 0.1 M KC1. Protein was prepared by homogenizing t~ue in 2 % NaC1. After centrifugation for 15 rain at 10,000 g, protein was precipitated by boiling the supernatant for 30 rain. The precipitate was dried by 1yophilization. Other types of compounds, e.g. organic acids, present in small amounts in potato, were isolated by ion exchange chromatography as described by Romberger and Norton (4). Compounds adsorbed by the cation and anion exchange resins, respec- tively, are amino acids and organic acids. The neutral fraction which passes through both resins is composed primarily of sugars. Amberlite or Dowex as exchange resins yielded similar results. All extracted substrates were converted to CO2 for mass spectrometry (8). THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 55 " 1970 Mass Spectrometer Analysis The 1~C/12C ratio in any given sample was com- pared with a standard. The standard used was the COs from the fossil carbonate skele- ton of Belemnitella americana (PDB1). The function defining the values reported here is ~I$c per mil -- (13C/12C) sample -- (lSC/12C) standard X I000 (13C/12C) standard Thus, a sample with a ~ uC per rail of - 10 has a lsC/~2C ratio less than the standard by 10 per mil, or 1.0 %. For example, the lnC/12C ratio of atmospheric CO2 is smaller than that of the PDB1 standard by about 7 per rail. Therefore, the 6 lsC per rail of this COs is --7. The precision of measuring 6 lsC with the mass spectrometer is -4-0.1 per mil.